This invention relates to an apparatus for automated preparation of DNA samples that are to be analyzed with a DNA sequencer. More particularly, the invention relates to an automated apparatus with which fluorescence-labelled DNA samples can be prepared with the aid of amplification by thermal cycling, in particular, amplification by polymerase chain reaction (PCR).
A specified DNA region can be amplified at least about 105-fold by repeating DNA synthesis reactions using two primers bracketing that region and a DNA synthetase. This method called polymerase chain reaction (PCR) was developed by the American company Cetus in 1985. It was later modified to use thermostable Taq polymerase and has been established as an efficient PCR-based method. In 1987, another American company Perkin-Elmer and Cetus jointly developed an automated PCR apparatus which contributed to a marked growth of the use of PCR.
Details about the theory of PCR are given in many prior art references. According to Jikken Igaku (Experimental Medicine), Vol. 7, No. 2, pp. 14-18 (1989), the PCR is the sequence of the following three steps: (1) duplex DNA to which primers are joined is thermally denatured to single strands, (2) the primers are annealed and (3) the primed DNA is extended by Taq polymerase. The cycle is repeated several tens of times to amplify the desired DNA fragment.
In the conventional automated PCR apparatus, polymerase chain reaction has been carried out in small test tubes such as plastic Eppendorf tubes which are placed within a chamber that permits temperature control. By changing the temperature in this chamber, the temperature of the reaction mixtures in the Eppendorf tubes is cyclically changed to the levels necessary for (1) denaturation (ca. 94xc2x0 C.), (2) annealing (ca. 55xc2x0 C.) and (3) extension (ca. 72xc2x0 C.).
The conventional automated PCR apparatus can yield only PCR products and their purification, sequencing reaction (for processing the PCR products to prepare samples that can be analyzed with a DNA sequencer) and the purification of the sequencing reaction product cannot be accomplished without a separate apparatus or manual operations.
An object, therefore, of the invention is to provide an apparatus for automated preparation of DNA samples by which an entire process comprising the preparation of PCR products, their purification, sequencing reaction and the purification of the sequencing reaction product can be performed automatically.
This object of the invention can be attained by an apparatus for automated preparation of DNA samples which comprises a reactor for preparing DNA samples and adjacent thereto an enzyme supply section, a plate holding section, a nozzle sealing section and a cleaning tank section, and wherein plates are loaded onto or unloaded from said plate holding section by means of a transport robot.
Said reactor for preparing DNA samples is supported on a unidirectionally moving mechanism to be capable of moving up and down and comprises a plurality of hollow electroconductive nozzles, hollow syringes coupled to said nozzles and pistons inserted into said syringes, the top of each of said pistons having a piston head secured thereto such that it can move up and down independently of said reactor for preparing DNA samples, the intermediate portions of said electroconductive nozzles being encased in a housing having an opening on both sides, a cooling mechanism being provided adjacent one of said openings, and electroconductive boards being connected to the intermediate portions of the electroconductive nozzles within said housing and also connected to a power supply via conductors.